Many human diseases and conditions are caused by gene mutations. Substantial effort has been directed towards the creation of transgenic animal models of such diseases and conditions to facilitate the testing of approaches to treatment, as well as to gain a better understanding of disease pathology. Early transgenic animal technology focused on the mouse, while more recent efforts, which have been bolstered by the development of somatic cell nuclear transfer (SCNT), have included larger animals, including pigs, cows, and goats. This technology has resulted in the production of, for example, pigs in which the gene encoding α-1,3-galactosyltransferase has been knocked out, in efforts to generate organs that can be used in xenotransplantation (see, e.g., Lai et al., Science 295:1089-1092, 2002). Further, this technology has resulted in the production of CFTR−/− and CFTR-ΔF508/ΔF508 pigs (see, e.g., U.S. Pat. No. 7,989,675 and U.S. patent application Ser. No. 12/283,980). Additional applications of this technology include the production of large quantities of human proteins (e.g., therapeutic antibodies; see, e.g., Grosse-Hovest et al., Proc. Natl. Acad. Sci. U.S.A. 101(18):6858-6863, 2004). Substantial benefits may be obtained by the use of somatic cell nuclear transfer technology in the production of large animal models of human disease.
One example of a condition caused in part by a genetic mutation is hypercholesterolemia. Hypercholesterolemia is a metabolic derangement indicated by the presence of high levels of cholesterol in the blood. Elevated cholesterol in the blood is due to abnormalities in the levels of lipoproteins, the particles that carry cholesterol in the bloodstream. Familial hypercholesterolemia results from mutations in the low-density lipoprotein receptor and can lead to premature or early onset atherosclerosis.
Thus, another example of a disease caused in part by a genetic mutation is atherosclerosis, also known as arteriosclerotic vascular disease or ASVD. As stated above, atherosclerosis results from longstanding elevation of serum cholesterol, a condition that is associated with hypercholesterolemia. Atherosclerosis is the primary cause of cardiovascular disease, which is the most common cause of death in the United States. See, e.g., Chart www.nhlbi.nih.gov/resources/docs/cht-book.htm. Atherosclerosis is characterized by the accumulation of lipids, cholesterol, calcium deposits, and cellular debris in vessel walls. This results in plaque formation, arterial obstruction, and diminished blood flow to organs. In time, these plaques can rupture and lead to thrombosis, resulting in myocardial infarction, stroke, or death. The main risk factors include elevated lipid levels, hypertension, and diabetes. While these factors are heavily influenced by diet and lifestyle, there are also genetic determinants that influence the disease.
More than 11 million Americans have atherosclerosis. Current treatment strategies for atherosclerosis are directed at changing patient lifestyle and/or diet and decreasing cholesterol in a patient via pharmacological methods. Surgical interventions such as balloon angioplasty and stent placement are used for advanced cases of the disease. While these therapeutic approaches have benefited many patients with this disease, they are far from ideal.
As the baby boomer generation ages, the American Heart Association projects heart disease deaths to increase 2.5 times faster than the population, and the prevalence of heart disease is projected to increase by 16% each decade. See, e.g., Lloyd-Jones, D et al., Circulation 121 (7), 948-54, 2010. The estimated annual financial impact of cardiovascular disease in the U.S. today is $475 billion. Pharmaceutical companies invest over $8 billion per year in cardiovascular research, and federal funding accounts for an additional $2 billion annually. Yet, despite these significant expenditures, current treatments remain inadequate because therapeutic strategies that show promise in the current model systems fail to yield results in patients. See, e.g., Hackam, D. G. et al., JAMA, 296 (14), 1731-2, 2006. This lack of predictive efficacy in the drug development process is costly, with over 70% of all drug development costs being the result of failed drugs.
Drug development using inappropriate animal models and translating those therapies to patients is inefficient and extremely costly. The lack of an appropriate animal model that accurately replicates one or more of the manifestations of human atherosclerosis has been a major barrier to the development of effective therapies, interventions, and diagnostic tools for this deadly disease. Several mouse models have been generated with mutations in genes important for lipoprotein metabolism. While these models have been informative, they fall short of being reliable predictive models for the study of atherosclerosis in humans because, among other things, mice fail to develop the complex atherosclerotic lesions that are typical of the human disease.
Atherosclerosis and hypercholesterolemia are associated with elevated low-density lipoprotein (LDL), which results, in part, from mutations in the low-density lipoprotein receptor (LDLR). The LDLR is a cell-surface glycoprotein found mainly in the liver that plays an important role in maintaining proper homeostasis of blood cholesterol. Upon ligand binding, the receptor-lipoprotein complex is endocytosed and trafficked to the endosome where an acidic environment causes the release of the LDL. The empty receptor is then recycled to the cell surface. The LDL is metabolized and excess cholesterol is excreted.
In contrast to mice, the physiology and anatomy of the porcine cardiovascular system closely resembles that of humans. In fact, pigs have been used as models of cardiovascular disease, and pigs with naturally occurring mutations in their LDLR gene, and therefore possessing elevated LDL, have been reported. See, e.g., Grunwald, K. A et al., J Lipid Res 40 (3), 475-85, 1999; Hasler-Rapacz, J et al., Am J Med Genet 76 (5), 379-86, 1998; Rapacz, J et al., Science 234 (4783), 1573-7, 1986. These naturally occurring mutations in the LDL receptor do not destroy its function; rather they decrease binding affinity for plasma LDL. Even on a normal diet, these pigs develop severe cardiovascular disease and provide the first animal model to develop spontaneous hypercholesterolemia and atherosclerotic lesions ranging from fatty streaks to advanced plaques, with accompanying calcification, hemorrhage, and rupture.
Although pigs with naturally occurring mutations in the LDLR gene are an attractive model for hypercholesterolemia and atherosclerosis, there are significant drawbacks. First, there has been substantial variability in the plasma cholesterol levels and disease development. This is likely due to the mild nature of the mutation and the broad, uncharacterized genetic background of these animals. In contrast, pigs with genetically engineered mutations in the LDLR gene, such as “null” mutations (for example, LDLR +/− and LDLR −/−), or any of the more than 1000 LDLR mutations identified in humans, will provide a much more consistent, predictable and reliable model for disease progression and development. Using the techniques described in the present invention, any one or more of the mutations in the LDLR gene could be created and studied in a large animal model. Second, the limited availability of pigs with a natural occurring mutation in the LDLR gene has prevented broader access by the research community. Finally, because the pronounced atherosclerotic lesions typically take 3-4 years to develop and these pigs are from a very large, domestic pig breed, the study of these animals is difficult and expensive.
A large animal model that accurately replicates the manifestations of human hypercholesterolemia and atherosclerosis and shares similarities to humans in size, anatomy, physiology, and genetics would be a transformative resource in bridging the substantial gap between models currently used for early-stage drug discovery and Phase 0/I human clinical trials. Furthermore, there is great interest in advancing medical devices, interventional strategies, and non-invasive diagnostic methods beyond their current state, but these fields are also limited by the current models systems. Rodent models are not well suited for most of these applications due to their size, and domestic pigs fed high cholesterol diets are excessively large, difficult to house and handle, and suffer from significant variability in phenotype. See, e.g., Daugherty, A., Mouse models of atherosclerosis. Am J Med Sci, 323 (1), 3-10 (2002). An LDLR-deficient miniature pig would benefit multiple disciplines within the cardiovascular disease community. Therefore, in one aspect of the invention, the transgenic animal model is a new model for hypercholesterolemia and atherosclerosis in a miniature pig breed. In one embodiment, the present invention accomplishes this in two steps by combining gene targeting and SCNT.